This invention relates to methods for treating inflammatory diseases by administration of a thrombin inhibitor.
Anti-inflammatory drugs include non steroidal anti-inflammatory drugs (NSAIDs) which exert anti-inflammatory, analgesic and antipyretic activity. These include salicylates such as aspirin, sodium salicylate, choline salicylate, salicylsalicylic acid, diflunisal, and salsalate; indoleacetic acids such as indomethacin and sulindac; pyrazoles such as phenylbutazone, oxyphenbutazone; pyrrolealkanoic acids such as tolmetin; phenylacetic acids such as ibuprofen, feroprofen, flurbiprofen, and ketoprofen; fenamates such as mefanamic acid, and meclofenamate; oxicams such as piroxicam; and naphthaleneacetic acids such as naproxen. Nearly all act by inhibiting cyclo-oxygenase activity. Aspirin, for example, acetylates and irreversibly inactivates cyclo-oxygenase. Others, such as indomethacin, inhibit cyclo-oxygenase activity reversibly by binding in a stereospecific manner to one or another subunit of the enzyme. NSAIDs are active in reducing the prostaglandin-induced pain and swelling associated with the inflammation process but are also active in affecting other prostaglandin-regulated processes not associated with the inflammation process. Thus, use of high doses of most common NSAIDs can produce severe side effects, including life threatening ulcers, that limit their therapeutic potential.
Adrenal corticosteroids, which are alternatives to NSAIDs for treating inflammatory diseases, have even more drastic side effects, especially when long term therapy is involved. These steroids, including hydrocortisone, prednisolone, 6 alpha-methylprednisolone, triamcinolone, dexamethasone and betaroethasone, affect inflammation by a possible mechanism whereby they bind to intracellular glucocorticoid receptors to subsequently initiate a series of cellular events involving synthesis of new phospholipid inhibitory proteins, or lipocortins, that can affect the inflammatory and the teratogenic responses of certain cells exposed to glucocorticoids. The anti-inflammatory effect of glucocorticoids has been well documented.
Excessive bleeding disorders are associated with development of inflammatory conditions. Hemophilia, a bleeding disorder caused by a deficiency clotting Factor VIII or clotting Factor IX, can result in recurring bleeding into joints and muscles that causes crippling inflammation and deformities. Hemophilia also causes swelling of the base of the tongue until it blocks the airway.
Thrombin inhibitors are known to be useful for treating or preventing venous thromboembolism (e.g. obstruction or occlusion of a vein by a detached thrombus; obstruction or occlusion of a lung artery by a detached thrombus), cardiogenic thromboembolism (e.g. obstruction or occlusion of the heart by a detached thrombus), arterial thrombosis (e.g. formation of a thrombus within an artery that may cause infarction of tissue supplied by the artery), atherosclerosis (e.g. arteriosclerosis characterized by irregularly distributed lipid deposits) in mammals, and for lowering the propensity of devices that come into contact with blood to clot blood, but have not been recognized as useful for treating inflammatory diseases.
As described below, it has now been found that thrombin inhibitors, which inhibit formation of blood clots, are in fact effective for inhibiting inflammation.
The invention is a method for treating an inflammatory disease in a patient which comprises treating the patient with a composition comprising a thrombin inhibitor. Such diseases include but are not limited to nephritis, systemic lupus erythematosus, rheumatoid arthritis, glomerulonephritis, and sacoidosis. In one class of the method, the thrombin inhibitor is selected from the group consisting of 3-(2-phenylethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylene-carboxamindomethylpyridinyl)-2-pyrazinone, Nxe2x80x2-[[1-(Aminoiminomethyl)-4-piperidinyl]methyl]-N-(3,3-diphenylpropionyl)-L-proline amide, and 3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylenecarboxamidomethylpyridinyl)-2-pyridinone or a pharmaceutically acceptable salt thereof.
The invention is also a method for treating an inflammatory disease in a patient which comprises treating the patient with a combination comprising a thrombin inhibitor and an NSAID, e.g., a COX-2 inhibitor. Such diseases include but are not limited to nephritis, systemic lupus, erythematosus, rheumatoid arthritis, glomerulonephritis, vasculitis and sacoidosis. In one class of the method, the thrombin inhibitor is selected from the group consisting of 3-(2-phenylethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylene-carboxamidomethylpyridinyl)-2-pyrazinone, Nxe2x80x2-[[1-(Aminoiminomethyl)-4-piperidinyl]methyl]-N-(3,3-diphenylpropionyl)-L-proline armide, and 3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylenecarboxamidomethylpyridinyl)-2-pyridinone or a pharmaceutically acceptable salt thereof and the COX-2 inhibitor is selected from the group consisting of 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone and 3-(3,4-difluorophenyl)4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone, or a pharmaceutically acceptable salt thereof.
The invention is also a method for inhibiting secondary inflammation in a patient developing inflammation at a primary site which comprises treating the patient with a composition comprising a thrombin inhibitor. Such inhibited secondary inflammation is inflammation that would otherwise occur in an untreated patient resulting from inflammatory diseases in which fibrin serves as a matrix onto which inflammatory cells migrate and adhere
The present invention is a method for relieving pain, fever and inflammation of a variety of conditions including nephritis, systemic lupus erythematosus, rheumatoid arthritis, glomerulonephritis, sacoidosis, rheumatic fever, symptoms associated with influenza or other viral infections, common cold, low back and neck pain, dysmenorrhea, headache, toothache, sprains and strains, myositis, neuralgia, synovitis, arthritis, including rheumatoid arthritis degenerative joint diseases (osteoarthritis), gout and ankylosing spondylitis, bursitis, burns, injuries, following surgical and dental procedures in a patient by administering to the patient a therapeutically effective amount of a thrombin inhibitor. Thrombin inhibitors may also be useful for the treatment of dementia including pre-senile and senile dementia, and in particular, dementia associated with Alzheimer Disease.
In inflammatory diseases wherein fibrin formation is prominent, the fibrin may be a determinant of the pathology. Fibrin serves as a matrix onto which inflammatory cells can migrate and adhere. (see Sherman et al., 1977 J. Exp. Med. 145:76-85; Altieri et al., 1986 J. Clin. Invest. 78:968-976; Wright et al., 1983 Proc. Natl. Acad. Sci. 85:7734-7738; Altieri et al., 1993 J. Biol. Chem. 268;1847-1853). Fibrin also enhances expression of the inflammatory cytokine EL-1beta and decreases expression of IL-1 receptor antagonist by human peripheral blood mononuclear cells (see Perez 1995 J. Immunol. 154:1879-1887). The anticoagulants warfarin and heparin attenuate delayed-type hypersensitivity reactions and experimental nephritis in animals. (see Jasain et al., Immunopathogenesis of Rheumatoid Arthritis Eds. G. S. Panayi et al., Surrey, UK, Reedbooks, Ltd. and Halpern et al., 1965 Nature 205:257-259). Enzymatic defibrination with ancrod diminishes the degree of experimental nephritis (Naish et al., 1972 Clin. Sci. 42:643-646), systemic lupus erythematosus (Cole et al., 1990 Kidney Int. 37:29-35, and rheumatoid arthritis (see Busso et al., 1998 J. Clin. Invest. 102:41-50) in animals, and glomerulonephritis in man (see Kim et al., 1988 Q. J. Med. 69:879-905). Additionally, intra articular injection of fibrin induces arthritis in rabbits immunized with fibrin Dumonde et al., 1961 British Journal of Experimental Pathology XLIII:373-383), and antigen-induced arthritis in mice is exacerbated in urokinase-deficient mice wherein fibrinolysis synovial fibrin is compromised (see Busso et al., 1998 J. Clin. Invest. 102:41-50).
In diseases where fibrin deposition is prominent such as, but not limited to, rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, vasculitis and sacoidosis, lowering the steady state concentration of fibrin by administration of a direct thrombin inhibitor will, according to the instant invention, diminish the pathological inflammatory responses associated with these diseases.
Thrombin Inhibitors
Thrombin inhibitors are compounds which inhibit hydrolytic activity of thrombin, including the catalytic conversion of fibrinogen to fibrin, activation of Factor V to Va, Factor VIII to VIIIa, Factor XIII to XIIIa, and activation of platelets.
Compounds may be identified as thrombin inhibitors by evaluating the compounds in assays described in S. D. Lewis et al., Thrombosis Research 70 pp. 173-190 (1993). One assay involves the measurement of rates of substrate hydrolysis, and the other involves measurement of activated partial thromboplastin time.
Hydrolysis assays may be carried out at 25xc2x0 C. in 0.05 M TRIS buffer pH 7.4, 0.15 M NaCl, 0.1% PEG. Trypsin assays may also contain 1 mM CaCl2. In assays wherein rates of hydrolysis of a p-nitroanilide (pna) substrate are determined, a Thermomax 96-well plate reader is used to measure (at 405 nm) the time dependent appearance of p-nitroaniline. sar-PR-pna is used to assay human xcex1-thrombin (Km=125 xcexcM) and bovine trypsin (Km=125 xcexcM). p-Nitroanilide substrate concentration is determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cmxe2x88x921Mxe2x88x921.
In certain studies with potent inhibitors (Ki less than 10 nM) where the degree of inhibition of thrombin is high, a more sensitive activity assay may be used. In this assay, the rate of thrombin catalyzed hydrolysis of the fluorogenic substrate Z-GPR-afc (Km=27 xcexcM) is determined from the increase in fluorescence at 500 nm (excitation at 400 nm) associated with production of 7-amino-4-trifluoromethyl coumarin. Concentrations of stock solutions of Z-GPR-afc are determined from measurements of absorbance at 380 nm of the 7-amino-4-trifluoromethyl coumarin produced upon complete hydrolysis of an aliquot of the stock solution by thrombin.
Activity assays are performed by diluting a stock solution of substrate at least tenfold to a final concentration xe2x89xa60.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor are determined in control experiments. Initial velocities of product formation in the absence (Vo) or presence of inhibitor (Vi) are measured. Assuming competitive inhibition, and that unity is negligible, Km/[S], [I]/e, and [I]/e (where [S], [I], and e respectively represent the total concentrations, of substrate, inhibitor and enzyme), the equilibrium constant (Ki) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of Vo/Vi on [I] shown in equation 1.
Vo/Vi=1+[I]/Kixe2x80x83xe2x80x83(1)
The activities shown by this assay indicate that the compounds of the invention are therapeutically useful for treating various conditions in patients suffering from unstable angina, refractory angina, myocardial infarction, transient ischernic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis, disseminated intravascular coagulation, and reocclusion or restenosis of recanalized vessels. The compounds of the invention are selective compounds, as evidenced by their inhibitory activity against human trypsin (represented by Ki), which is at least 1000 nM.
The anticoagulant effect of thrombin inhibitors can be verified according to the activated partial thromboplastin time assay described by S. D. Lewis et al., Thrombosis Research 70 pp. 173-190 (1993). According to the assay, citrated plasma is mixed with the test compound and with activated cephaloplastin reagent. Calcium chloride is then added to the mixture, and the degree of coagulation is measured. The absence of coagulation demonstrates the inability of the test compound to inhibit thrombin.
Thrombin inhibitors suitable for the present invention include those which inhibit thrombosis, including but not limited to those described in U.S. Pat. Nos. 5,536,708, 5,510,369, 5,672,582, 5,714,485, 5,629,324 (e.g. Nxe2x80x2-[[1-(Aminoiminomethyl)4-piperidinyl]methyl]-N-(3,3-diphenylpropionyl)-L-proline amide), U.S. Pat. No. 5,668,289 (e.g. 3-(2-Phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylenecarboxamidomethylpyridinyl)-2-pyridinone), U.S. Pat. Nos. 5,744,486, 5,798,377, WO9631504, WO09611941, WO09606832, WO09606849, WO09420467, WO9632110, U.S. Pat. No. 4,496,653, WO9715190, and WO9740024, e.g. 3-(2-Phenylethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylene-carboxamidomethylpyridinyl)-2-pyrazinone, the contents of which are hereby incorporated by reference. Such compounds are known to be useful for treating or preventing venous thromboembolism (e.g. obstruction or occlusion of a vein by a detached thrombus; obstruction or occlusion of a lung artery by a detached thrombus), cardiogenic thromboembolism (e.g. obstruction or occlusion of the heart by a detached thrombus), arterial thrombosis (e.g. formation of a thrombus within an artery that may cause infarction of tissue supplied by the artery), atherosclerosis (e.g. arteriosclerosis characterized by irregularly distributed lipid deposits) in mammals, and for lowering the propensity of devices that come into contact with blood to clot blood.
The thrombin inhibitors of the invention can be administered in such oral forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixers, tinctures, suspensions, syrups, and emulsions. Likewise, they may be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as an anti-aggregation agent. For treating ocular build up of fibrin, the compounds may be administered intraocularly or topically as well as orally or parenterally.
The thrombin inhibitors can be administered in the form of a depot injection or implant preparation which may be formulated in such a manner as to permit a sustained release of the active ingredient. The active ingredient can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as depot injections or implants. Implants may employ inert materials such as biodegradable polymers or synthetic silicones, for example, Silastic, silicone rubber or other polymers manufactured by the Dow-Corning Corporation.
The thrombin inhibitors can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearyl aamine or phosphatidylcholines.
The thrombin inhibitors may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The thrombin inhibitors may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinlypyrrolidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the thrombin inhibitors may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels.
The dosage regimen utilizing the thrombin inhibitors is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.
Oral dosages of the thrombin inhibitors, when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 30 mg/kg/day, preferably 0.025-7.5 mg/kg/day, more preferably 0.1-2.5 mg/kg/day, and most preferably 0.1-1.0 mg/kg/day (unless specified otherwise, amounts of active ingredients are on free base basis). For example, an 80 kg patient would receive between about 0.8 mg/day and 2.4 g/day, preferably 2-600 mg/day, more preferably 8-200 mg/day, and most preferably 8-80 mg/day. A suitably prepared medicament for once a day administration would thus contain between 0.8 mg and 2.4 g, preferably between 2 mg and 600 mg, more preferably between 8 mg and 200 mg, and most preferably 8 mg and 80 mg, e.g., 8 mg, 20 mg, 40 mg and 80 mg. Advantageously, the thrombin inhibitors may be administered in divided doses of two, three, or four times daily. For administration twice a day, a suitably prepared medicament would contain between 0.4 mg and 4 g, preferably between 1 mg and 300 mg, more preferably between 4 mg and 100 mg, and most preferably 4 mg and 40 mg, e.g., 4 mg, 10 mg, 20 mg and 40 mg.
Intravenously or subcutaneously, the patient would receive the active ingredient in quantities sufficient to deliver between 0.025-7.5 mg/kg/day, preferably 0.1-2.5 mg/kg/day, and more preferably 0.1-1.0 mg/kg/day. Such quantities may be administered in a number of suitable ways, e.g. large volumes of low concentrations of active ingredient during one extended period of time or several times a day, low volumes of high concentrations of active ingredient during a short period of time, e.g. once a day. Typically, a conventional intravenous formulation may be prepared which contains a concentration of active ingredient of between about 0.01-1.0 mg/ml, e.g. 0.1 mg/ml, 0.3 mg/ml, and 0.6 mg/ml, and administered in amounts per day of between 0.01 ml/kg patient weight and 10.0 ml/kg patient weight, e.g. 0.1 ml/kg, 0.2 ml/kg, 0.5 ml/kg. In one example, an 80 kg patient, receiving 8 ml twice a day of an intravenous formulation having a concentration of active ingredient of 0.5 mg/ml, receives 8 mg of active ingredient per day. Glucuronic acid, L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration may be used as buffers. Consideration should be given to the solubility and chemical compatibility of the drug in choosing an appropriate excipient. Subcutaneous formulations, preferably prepared according to procedures well known in the art at a pH in the range between 7.0 and 7.4, also include suitable buffers and isotonicity agents. They are formulated to deliver a daily dose of thrombin inhibitor in one or more daily subcutaneous administrations, e.g., one, two or three time each day. The choice of appropriate buffer and pH of a formulation, depending on solubility of the drug to be administered, is readily made by a person having ordinary skill in the art.
The compounds can also be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime.
The thrombin inhibitors are typically administered as active ingredients in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as xe2x80x9ccarrierxe2x80x9d materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixers, syrups and the like, and consistent with convention pharmaceutical practices.
For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn-sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch methyl cellulose, agar, bentonite, xanthan gum and the like.
Typical uncoated tablet cores suitable for administration of thrombin inhibitors are comprised of, but not limited to, the following amounts of standard ingredients:
Mannitol, microcrystalline cellulose and magnesium stearate may be substituted with alternative pharmaceutically acceptable excipients.
All of the thrombin inhibitor, cellulose, and a portion of the corn starch are mixed and granulated to 10% corn starch paste. The resulting granulation is sieved, dried and blended with the remainder of the corn starch and the magnesium stearate. The resulting granulation is then compressed into tablets containing 25.0, 50.0, and 100.0 mg, respectively, of active ingredient per tablet.